Highly active antiretroviral therapy (HAART) has had a major impact on the AIDS epidemic in industrially advanced nations; however, no eradication of human immunodeficiency virus type 1 (HIV-1) appears to be currently possible, in part due to the viral reservoirs remaining in blood and infected tissues. Moreover, we have encountered a number of challenges in bringing about the optimal benefits of the currently available therapeutics of AIDS and HIV-1 infection to individuals receiving HAART. They include (i) drug-related toxicities; (ii) partial restoration of immunologic functions once individuals developed AIDS; (iii) development of various cancers as a consequence of survival prolongation; (iv) flaming-up of inflammation in individuals receiving HAART or immune re-construction syndrome (IRS); and (v) increased cost of antiviral therapy. Such limitations and flaws of HAART are exacerbated by the development of drug-resistant HIV-1 variants. Successful antiviral drugs, in theory, exert their virus-specific effects by interacting with viral receptors, virally encoded enzymes, viral structural components, viral genes, or their transcripts without disturbing cellular metabolism or function. However, at present, no antiretroviral drugs or agents are likely to be completely specific for HIV-1 or to be devoid of toxicity or side effects in the therapy of AIDS, which has been a critical issue because patients with AIDS and its related diseases will have to receive antiretroviral therapy for a long period of time, perhaps for the rest of their lives. Thus, the identification of new class of antiretroviral drugs which have an unique mechanism(s) of action and produce no or minimal side effects remains an important therapeutic objective. We previously identified a new class of nucleoside reverse transcriptase inhibitors (NRTIs), 4-ethynyl-2-deoxunucleoside analogs (EdNs), which, unlike conventional NRTIs such as azidothymidine, contain the 3-hydroxy moiety. These EdNs exert potent antiviral activity agains a wide spectrum of HIV-1 and HIV-2 strains. In the time period of the annual report, we examined the intracytoplasmic anabolism and kinetics of antiviral activity against HIV-1 of a nucleoside reverse transcriptase inhibitor, 4-ethynyl-2-fluoro-2-deoxyadenosine (EFdA), which has a potent activity against various HIV-1 strains including multi-drug resistant HIV-1 variants. When CEM cells were exposed to 0.1 microM 3H-labled EFdA (3H-EFdA) or 3H-labeled-3-azido-2,3-dideoxythymidine (3H-AZT) for 6 hours, the intracellular amount of triphosphates (TP) of EFdA was 91.6 pmol/109 cells, while that of AZT was 396.5 pmol/109 cells. When CEM cells were exposed to a higher concentration of 3H-EFdA (10 microM), the amounts of EFdA-TP substantially increased by 22-fold (2,090 pmol/109 cells), while the amount of 3H-AZT-TP only moderately increased by 2.4-fold (970 pmol/109 cells). The intracellular T1/2 values of EFdA-TP and AZT-TP were approximately 17 and 3 hours, respectively. When MT-4 cells were cultured with 0.01 microM EFdA for 24 hours, thoroughly washed, exposed to HIV-1NL4-3 following incubation without EFdA, and further cultured for an additional 5 days, the values of % protection against HIV-1NL4-3 were 75 and 47 for the cells exposed to HIV-1NL4-3 following 24 and 48 hours no-drug incubation, while those with 1 microM AZT were 55 and 9.2, respectively. The IC50 values of EFdA-TP against human polymerases alpha, beta and gamma were more than 100, more than 100, and 10 microM, while those of ddA-TP were more than 100, 0.2, and 0.2 microM, respectively. These data, taken together, suggest that EFdA might represent a potent anti-HIV-1 agent with a possibility of once- or twice-a-day regimen and warrant further development as a potential therapeutic for those harboring wild-type HIV-1 and/or multi-drug resistant variants. In another area, we have been focusing on the design and synthesis of non-peptidyl protease inhibitors (PIs) that are potent against HIV-1 variants resistant to the currently approved PIs. One such anti-HIV-1 agent, darunavir (DRV)/TMC114 containing a structure-based designed privileged nonpeptidic P2 ligand, 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF), has recently been approved as a therapeutic agent for the treatment of individuals who harbor multi-drug-resistant HIV-1 variants and do not respond to previously existing HAART regimens. We recently designed, synthesized, and identified GRL-98065, a novel non-peptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing the structure-based designed privileged cyclic ether-derived non-peptide P2 ligand, 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF), and a sulfonamide isostere, which is highly potent against laboratory HIV-1 strains and primary clinical isolates (IC50: 0.0002-0.0005 microM) with minimal cytotoxicity (CC50: 35.7 microM in CD4+ MT-2 cells). GRL-98065 blocked the infectivity and replication of each of HIV-1NL4-3 variants exposed to and selected by up to 5 microM concentration of saquinavir, indinavir, nelfinavir, or ritonavir, and 1 microM concentration of lopinavir, or atazanavir (IC50: 0.0015 - 0.0075 microM), although it was less active against HIV-1NL4-3 selected by amprenavir (IC50: 0.032 microM). GRL-98065 was also potent against multi-PI-resistant clinical HIV-1 variants isolated from patients who had no response to existing antiviral regimens after having received a variety of antiviral agents, HIV-1 isolates of various subtypes, as well as HIV-2 isolates examined. Structural analyses revealed that the close contact of GRL-98065 with the main chain of the protease active site amino acids (Asp-29 and Asp-30) is important for its potency and wide-spectrum activity against multi-PI-resistant HIV-1 variants. The present data demonstrate that the privileged non-peptide P2 ligand, bis-THF, is critical for the binding of GRL-98065 to the HIV protease substrate binding site and this scaffold can confer highly potent antiviral activity against a wide spectrum of HIV isolates.